Direct measurement of mavacamten and deoxyATP perturbation of the SRX/DRX ratio in porcine cardiac myofibrils using a simple, accessible and multiplexed approach

Figures

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  Fig. 1. Overview of the method for rapid multiplexed imaging of myofibrils. (a) Myofibrils are purified from left ventricular cardiac trabeculae and then injected into a microfluidic cell. After wash-in and then wash-out of Cy3-ATP, the bulk fluorescence over the myofibril is measured and shows a decay over the 30-minute imaging period. (b) The fluorescence decay is best fitted as shown to a triple exponential. This is determined by examination of the (c) residuals shown for single (blue), double (orange) and triple (green) exponential fits. The single exponential fit residuals are plotted against the right Y-axis showing the large differences from the data. For the double and triple exponentials, these are plotted against the left Y-axis scale. The triple exponential fit (green) is preferred because it shows a better fit throughout the time range particularly at the longer time periods where it is important to obtain a good fit. All data were fit using a logarithmic weighting (due to their exponential distribution), therefore the residuals do not average on zero as would be expected from linear weighting

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  Fig. 2. Perturbing the DRX/SRX ratio using mavacamten and dATP. (a) Untreated data fitted to a Gaussian mixtures model shows two major populations. The colour distribution reflects the probability density of the 2D Gaussian used for fitting and the surrounding black circle is the density at 99.95% probability; this was used to independently define the boundaries between the populations for the SRX vs. DRX populations. (b) Rate constants for all myofibrils are plotted versus their percent amplitude (taking only the slower two phases of the transients (see Fig. 1b)). Values with only a single population are marked at 100%. This representation provides an overview of all the data, showing untreated (filled circles), treated with 30 µM mavacamten (squares) and 100% dATP (open circles). The vertical line in the graph represents the boundary rate constant defined by the Gaussian mixtures model. For clarity, (a) and (b) are aligned. (c) Provides a percentage of the DRX/SRX populations determined from the fitted data for each myofibril averaged across all measured myofibrils. This chart shows relative to DRX in untreated 68.0% (± 3.2 SEM n = 10 myofibrils) there is a significant decrease (p <…